This PDF file contains the front matter associated with SPIE Proceedings Volume 10414 including the Title Page, Copyright information, Table of Contents, Introduction, and Conference Committee listing.

A region of interest (ROI) based quantitative FLIM-FRET image analysis is developed to quantitate the autofluorescence signals of the essential amino acid tryptophan as a biomarker to investigate the metabolism in prostate cancer cells.

Newly developed microscopy methods have the goal to give researches in bio-molecular science a better understanding of processes ongoing on a cellular level. Especially two-photon excited fluorescence (TPEF) microscopy is a readily applied and widespread modality. Compared to one photon fluorescence imaging, it is possible to image not only the surface but also deeper lying structures. Together with fluorescence lifetime imaging (FLIM), which provides information on the chemical composition of a specimen, deeper insights on a molecular level can be gained. However, the need for elaborate light sources for TPEF and speed limitations for FLIM hinder an even wider application. In this contribution, we present a way to overcome this limitations by combining a robust and inexpensive fiber laser for nonlinear excitation with a fast analog digitization method for rapid FLIM imaging. The applied sub nanosecond pulsed laser source is perfectly suited for fiber delivery as typically limiting non-linear effects like self-phase or cross-phase modulation (SPM, XPM) are negligible. Furthermore, compared to the typically applied femtosecond pulses, our longer pulses produce much more fluorescence photons per single shot. In this paper, we show that this higher number of fluorescence photons per pulse combined with a high analog bandwidth detection makes it possible to not only use a single pulse per pixel for TPEF imaging but also to resolve the exponential time decay for FLIM. To evaluate our system, we acquired FLIM images of a dye solution with single exponential behavior to assess the accuracy of our lifetime determination and also FLIM images of a plant stem at a pixel rate of 1 MHz to show the speed performance of our single pulse two-photon FLIM (SP-FLIM) system.

A polarimetric third-harmonic generation microscopy based on nonlinear Stokes-Mueller polarimetry theory is presented. All eight observable χ⁽³⁾ tensor components of a β-carotene crystallite are extracted and a chiral component is identified.

We combined Second Harmonic Generation, Two-Photon Fluorescence and Fluorescence Lifetime Imaging Microscopy for studying human carotid ex vivo tissue sections affected by atherosclerosis, resulting in the discrimination of different arterial regions within the plaques.

Third Harmonic Generation (THG) imaging was applied to mouse resting and activated T-cells. Quantification of THG signal, which corresponded to lipid droplets, could distinguish activated Tcells, allowing follow-up of immune response development.

SHG microscopy was successfully used to characterize the orientation of sutural lamellae within corneal samples by means of an epi-detection scheme. Sutural lamellae are located in the first 30 μm of the stromal layer below Bowman’s membrane; they affect the mechanical properties of the cornea and are responsible of the overall corneal stiffness. This stromal layer is particularly interesting for diagnosing and following-up keratoconus, an eye disorder that features a reduced stiffness of the cornea and its consequent pathological deformation. The methodology was tested by acquiring image stacks of the central portion of the cornea in ex vivo samples of healthy corneas, keratoconic corneas and keratoconic corneas treated with cross-linking (CXL), a treatment aimed at hindering the progression of keratoconus. The samples were imaged and characterized based on forward/backward SHG ratio, finding a different ratio in keratoconus with respect to both the healthy cornea and cross-linked keratoconus. The inclination of corneal sutural lamellae was then characterized by means of a three-dimensional correlation analysis on SHG images, acquired using a backward detection geometry. Such method provided good discrimination capabilities, demonstrating that this approach can be used not only for diagnosing keratoconus in a very early stage, but also for performing treatment follow-up on cross-linked corneas.

Modern biomedical imaging modalities aim to provide researchers a multimodal contrast for a deeper insight into a specimen under investigation. A very promising technique is stimulated Raman scattering (SRS) microscopy, which can unveil the chemical composition of a sample with a very high specificity. Although the signal intensities are enhanced manifold to achieve a faster acquisition of images if compared to standard Raman microscopy, there is a trade-off between specificity and acquisition speed. Commonly used SRS concepts either probe only very few Raman transitions as the tuning of the applied laser sources is complicated or record whole spectra with a spectrometer based setup. While the first approach is fast, it reduces the specificity and the spectrometer approach records whole spectra -with energy differences where no Raman information is present-, which limits the acquisition speed. Therefore, we present a new approach based on the TICO-Raman concept, which we call sparse-sampling. The TICO-sparse-sampling setup is fully electronically controllable and allows probing of only the characteristic peaks of a Raman spectrum instead of always acquiring a whole spectrum. By reducing the spectral points to the relevant peaks, the acquisition time can be greatly reduced compared to a uniformly, equidistantly sampled Raman spectrum while the specificity and the signal to noise ratio (SNR) are maintained. Furthermore, all laser sources are completely fiber based. The synchronized detection enables a full resolution of the Raman signal, whereas the analogue and digital balancing allows shot noise limited detection. First imaging results with polystyrene (PS) and polymethylmethacrylate (PMMA) beads confirm the advantages of TICO sparse-sampling. We achieved a pixel dwell time as low as 35 μs for an image differentiating both species. The mechanical properties of the applied voice coil stage for scanning the sample currently limits even faster acquisition.

We use an ultrafast diode-pumped semiconductor disk laser (SDL) to demonstrate several applications in multiphoton microscopy. The ultrafast SDL is based on an optically pumped Vertical External Cavity Surface Emitting Laser (VECSEL) passively mode-locked with a semiconductor saturable absorber mirror (SESAM) and generates 170-fs pulses at a center wavelength of 1027 nm with a repetition rate of 1.63 GHz. We demonstrate the suitability of this laser for structural and functional multiphoton in vivo imaging in both Drosophila larvae and mice for a variety of fluorophores (including mKate2, tdTomato, Texas Red, OGB-1, and R-CaMP1.07) and for endogenous second-harmonic generation in muscle cell sarcomeres. We can demonstrate equivalent signal levels compared to a standard 80-MHz Ti:Sapphire laser when we increase the average power by a factor of 4.5 as predicted by theory. In addition, we compare the bleaching properties of both laser systems in fixed Drosophila larvae and find similar bleaching kinetics despite the large difference in pulse repetition rates. Our results highlight the great potential of ultrafast diode-pumped SDLs for creating a cost-efficient and compact alternative light source compared to standard Ti:Sapphire lasers for multiphoton imaging.

Based on the modal analysis method, a model describing the output beam of a diode pumped solid state (DPSS) laser emitting a multimode beam is presented. Measuring the output beam profiles in near-field, the individual modes as well as their contributions are determined. To this purpose, the superposition principle and the angular spectrum method are employed to predict the beam profile along the propagation axis and at far-field.

In this paper, we propose a differential single-pixel imaging (SPI) approach based on time stretch and demonstrate its capability for image quality improvement and reconstruction time reduction versus the conventional timestretch- based SPI approach.

We propose a new imaging platform based on lens-free time-lapse microscopy for 3D cell culture and its dedicated algorithm lying on a fully 3D regularized inverse problem approach. First 3D+t results are presented

This study clearly demonstrates that multispectral confocal microscopy images analyzed by artificial neural networks provides a powerful tool to real-time monitoring photosensitizer uptake, as well as photochemical transformations occurred.

We demonstrate photothermal quantitative phase imaging of living cells with functionalized nanoparticles in a cost-efficient setup and quantify the impact of nanoparticle excitation with low modulation frequencies on cell morphology and viability.

This paper reports an improvement in the development of a low-cost QPI microscope offering new capabilities in term of phase measurement accuracy for label-free live samples in the longer term (i.e., hours to days). The spatially separated scattered and non-scattered image light fields are reshaped in the Fourier plane and modulated to form an interference image at a CCD camera. The apertures that enable these two beams to be generated have been optimised by means of laser-cut apertures placed on the mirrors of a Michelson interferometer and has improved the phase measuring and reconstruction capability of the QPI microscope. The microscope was tested with transparent onion cells as an object of interest.

A spectrally resolved white light interferometry is demonstrated using a discrete spectrum light sources (i.e., RGB LEDs) and monochrome CCD camera for the multi-color quantitative phase imaging of biological cells without color cross talk.

A spatially low coherent light source is synthesized to reduce the spatial phase noise in the laser based digital holographic microscope, which otherwise introduces unwanted spatial phase, subsequently, the height measurement error of the biological objects.

We demonstrate results for phase maps of biological cells using white-light and multi-spectral interference microscopy. Study on comparison of phase maps reconstructed using 1-CCD and 3- CCD is presented to reduce color cross-talk and improved resolution.

Digital holographic microscopy (DHM) was used as multimodal optical method for nanomaterial toxicity testing that overcomes the limitations and assay disturbances of conventional in vitro assays based on absorbance or fluorescence read outs.

In digital holographic microscopy, one often obtains an in-focus image of the sample by applying a focus metric to a stack of numerical reconstructions. We present an alternative approach using a deep convolutional neural network.

The precise location and nature of the artery water permeability barrier is unknown. Water imaging in arteries with femtosecond coherent anti-Stokes Raman scattering microscopy reveals that this barrier is formed by the endothelial basolateral membrane.

Advanced methods of microscopy and advanced techniques of analysis stemming therefrom have developed greatly in the past few years.The use of single discrete methods has given way to the combination of methods which means an increase in data for processing to progress to the analysis and diagnosis of ailments and diseases which can be viewed by each and any method.This presentation shows the combination of such methods and gives example of the data which arises from each individual method and the combined methodology and suggests how such data can be streamlined to enable conclusions to be drawn about the particular biological and biochemical considerations that arise.In this particular project the subject of the methodology was human lactoferrin and the relation of the adhesion properties of hlf in the overcoming of barriers to adhesion mainly on the perimeter of the cellular unit and how this affects the process of immunity in any particular case.

Light-sheet microscopy enables whole mouse brain imaging in association with clearing methodologies. Here, we present a pipeline for optimal investigation of the vascular component, which offers improved image quality for morphological analysis.

Composite fibers of bioabsorbable poly-butylene adipate-co-terephthalate (PBAT) reinforced with superhydrophilic carbon nanotubes and hydroxyapatite nanocrystals were obtained by rotary jet spinning technique (RJS). The fibers were morphologically and biologically analyzed and found of potential use as scaffold for hard tissue engineering.

A miniaturized setup for sample rotation on a microscope stage has been developed, combined with light sheet, confocal or structured illumination microscopy and applied to living cells as well as to small organisms. This setup permits axial tomography with improved visualization of single cells or small cell clusters as well as an enhanced effective 3D resolution upon sample rotation.

We combined Second Harmonic Generation and Two-Photon Fluorescence for imaging ex vivo tissue sections of human bladder affected by urothelial carcinoma. We studied different grades of the tumor, and compared them to healthy bladder mucosa.

Single-pixel detection approaches have been applied with success in different imaging techniques such as optical microscopy. Here we show that it is possible to improve the resolution of single-pixel microscopy by using an array of photodetectors and Fourier ptychography algorithms.

During long-term imaging, cells move out of the field of view. We have generated functionalized substrates containing rectangular areas, which were capable in keeping cells over the whole observation period.

We present the combination of a single-shot water-immersion digital holographic microscopy with broadband illumination for simultaneous visualization of coherent and incoherent images using microbeads and different biosamples.

Measurements of average phase shifts introduced by living HeLa cells to probe wave front were carried out. Variations of this value were monitored in the course of morphological changes caused by photodynamic treatment at various irradiation doses. Observations of changes in living cells were also performed by means of far field optical microscopy and confocal fluorescent microscopy. Quantitative analysis of the data obtained shows that average phase shift introduced by the cells may either increase or decrease depending upon major parameters of the treatment.

In digital holographic interferometry (DHI), coherent noise degrades accuracy of phase information. We present multi-beam polarization DHI in which two cross polarized interferograms are recorded. Fourier analysis of interferograms reduces coherent noise and increases accuracy.

Adequate supply of oxygen to the body is the most essential requirement. In vertebrate species this function is performed by Hemoglobin contained in red blood cells. The mass concentration of the Hb determines the oxygen carrying capacity of the blood. Thus it becomes necessary to determine its concentration in the blood, which helps in monitoring the health of a person. If the amount of Hb crosses certain range, then it is considered critical. As the Hb constitutes upto 96% of red blood cells dry content, it would be interesting to examine various physical and mechanical parameters of RBCs which depends upon its concentration. Various diseases bring about significant variation in the amount of hemoglobin which may alter certain parameters of the RBC such as surface area, volume, membrane fluctuation etc. The study of the variations of these parameters may be helpful in determining Hb content which will reflect the state of health of a human body leading to disease diagnosis. Any increase or decrease in the amount of Hb will change the density and hence the optical thickness of the RBCs, which affects the cell membrane and thereby changing its mechanical and physical properties. Here we describe the use of lateral shearing digital holographic microscope for quantifying the cell parameters for studying the change in biophysical properties of cells due to variation in hemoglobin concentration.

We have developed a three-dimensional tracking system for microscopes. Using the astigmatic lens method and a CMOS image sensor, we realize a rapid detection of a target position in a wide range. We demonstrate a target tracking using the developed system.